Urinary tract infections (UTIs) are one of the most common types of bacterial infection encountered by humans throughout their lifespan and are a major health problem afflicting millions of people each year. Gram-negative bacteria cause the majority of infections of which Escherichia coli is the primary pathogen and prone to antibiotic resistance. The standard methods for species diagnosis and susceptibility determination are culture- based protocols that take up to 48 h. As a result, UTIs are one of the most frequent reasons for antimicrobial prescriptions in healthcare facilities, without the benefit of a confirmed diagnosis. Therefore, modern day rapid diagnostic methods that promote rapid identification and antimicrobial stewardship are crucial. The long-term objective of this R&D is to develop an easy to use platform for the rapid (?3 h) identification and antimicrobial susceptibility testing (ID/AST) of UTI pathogens directly from urine. A major goal is to develop a diagnostic that can bypass the need for bacterial amplification and isolation and thus overcome the major time-limiting step of current diagnostics. The platform will consist of species-specific bacteriophages (phages) engineered to express heterologous marker proteins, a lateral flow immunoassay, and analysis by Sofia, a fluorescent assay reader produced and marketed by our Quidel collaborators. Upon bacterial cell infection, the recombinant phages will produce large amounts of a foreign marker protein, which following phage-mediated cell lysis, can be rapidly and sensitively detected by an immunoassay and objectively read by Sofia. As the marker protein production is correlated to cell fitness, the platform termed Phage-accelerated Test (PhACT), can rapidly determine an isolates sensitivity, or resistance to a particular antibiotic, a key attribute in this 'post-antibiotic' era. The Phase I proof-of-principle studies will focus on demonstrating feasibility for E. coli as this species is responsible for >70% of uncomplicated UTIs. Specific Aim 1 will generate a cocktail of genetically engineered E. coli phages harboring the foreign marker protein. Specific Aim 2 will develop phage-mediated lateral-flow immunoassay and determine its performance characteristics for direct ID/AST from urine samples. The research is significant as the technology enables simultaneous detection and phenotypic drug susceptibility analysis without culture amplification and isolation, thus bypassing the major time limiting step in diagnostics. This is key as decreasing length of time for diagnosis and the administration of appropriate therapy positively impacts patient outcomes and promotes antimicrobial stewardship.